A changing pattern of brain-derived neurotrophic factor expression correlates with the rearrangement of fibers during cochlear development of rats and mice

Abstract

The reorganization of specific neuronal connections is a typical feature of the developing nervous system. It is assumed that the refinement of connections in sensory systems requires spontaneous activity before the onset of cochlear function and selective sensory experience during the ensuing period. The mechanism of refinement through sensory experience is currently postulated as being based on the selective reinforcement of active projections by neurotrophins. We studied a presumed role of neurotrophins for rearrangement of afferent and efferent fibers before the onset of sensory function in the precisely innervated auditory end organ, the cochlea. We observed a spatiotemporal change in the localization of brain-derived neurotrophic factor (BDNF) protein and mRNA, which correlated with the reorganization of fibers. Thus, BDNF decreased in target hair cells during fiber retraction and was subsequently upregulated in neurons, target hair cells, and adjacent supporting cells concomitant with the formation of new synaptic contacts. Analysis of the innervation pattern in BDNF gene-deleted mice by immunohistochemistry and confocal microscopy revealed a failure in the rearrangement of fibers and a BDNF dependency of distinct neuronal projections that reorganize in control animals. Our data suggest that, before the onset of auditory function, a spatiotemporal change in BDNF expression in sensory, epithelial, and neuronal cells may guide the initial steps of refinement of the innervation pattern.

Fig. 1.

Toluidine blue stained section of an organ of Corti of a mouse at P5, indicating the typical cellular details of an organ at that age. SC, Supporting cells;TM, tectorial membrane. Scale bar, 20 μm.

Fig. 2.

Localization of BDNF protein in spiral ganglion cells and organ of Corti in mice cochleae (BDNF +/+) at P1, P3, P6, and P10 by immunohistochemistry. In IHCs (arrowhead) and OHCs (vertical arrows) of the organ of Corti, BDNF was expressed at P1, was absent at P3, reappeared at P6, and was downregulated again at P10. At P6, BDNF was also detected in supporting cells as pillar cells (PC), Deiters’ cells (DC), and border cells (BC). In the spiral ganglion (SG), no BDNF staining was detected at P1, single neurons expressed BDNF at P3 (arrowhead), and the maximal number of immunopositive neuronal cell bodies was reached between P4 and P6 (P6, filled arrowheads). During this time, we observed BDNF in neuronal fibers (P6, open arrowheads). The experiment was repeated in triplicate with similar results. The staining of the tectorial membrane (TM) was nonspecific. Scale bar, 20 μm.

Fig. 3.

Characterization of anti-BDNF antibody.A, Cross-reactivity of anti-BDNF antibody with neurotrophins NT-3, NT-4, and NGF was tested in Western blot analysis. Anti-BDNF recognized only rhBDNF but not rhNT-3, rhNT-4, or rhNGF.B, Sensitivity of anti-BDNF antibody was tested in Western blot using indicated concentrations of BDNF. The minimal amount rhBDNF recognized by anti-BDNF antibody was 5 ng. C, In a Western blot with rat cochlear protein, anti-BDNF antibody recognized a protein (arrow) close to the molecular weight of rhBDNF, which is likely to be the mature BDNF protein. Anti-BDNF antibody also recognized proteins of ∼20 and ∼30 kDa, probably BDNF precursor proteins. D, Anti-BDNF immunoreaction (Anti-BDNF) in hair cells (arrowheads and vertical arrows) was completely abolished by preabsorption of the antibody with rhBDNF (Anti-BDNF+rhBDNF) but not with rhNT-3 (Anti-BDNF+rhNT-3). Immunoreaction in the tectorial membrane (TM) was nonspecific. Scale bar, 20 μm.

Fig. 4.

Localization of BDNF mRNA in the organ of Corti and the spiral ganglion of rats by in situhybridization. Although no BDNF mRNA was detected at P3, BDNF mRNA was localized in OHCs (vertical arrows), pillar cells (PC), Deiters’ cells (DC), and border cells (BC) of the organ of Corti at P6. At P8, BDNF mRNA was noted restrictively in Deiters’ and border cells. In the spiral ganglion (SG), BDNF hybridization signal in neurons was not effected by cohybridization with an excess of nonlabeled NT-3 antisense (BDNF+nlNT-3) but was completely abolished by cohybridization with an excess of nonlabeled BDNF antisense (BDNF+nlBDNF). Scale bars, 20 μm.

Fig. 5.

Localization of BDNF protein and synaptophysin protein in the midbasal cochlea turn of mice (BDNF +/+) by immunohistochemistry. The absence of BDNF protein in the organ of Corti at P2 (P2, BDNF) is associated with a detection of synaptophysin-immunopositive presynaptic specializations in OHC soma (P2,Synaptophysin). The appearance of BDNF in outer hair cells and supporting cells as pillar cells (PC), Deiters’ cells (DC), and border cells (BC) between P4 and P6 occurs parallel to the loss of synaptophysin-immunopositive presynaptic specializations in outer hair cells and the appearance of synaptophysin immunoreactivity in MOC efferents (P6, Synaptophysin). Scale bar, 10 μm.

Fig. 6.

Localization of presynaptic specializations in outer hair cells and efferents innervating outer hair cells in the midbasal cochlea turn in control and BDNF gene-deleted mice analyzed by immunohistochemistry using an anti-synaptophysin antibody. At P2, synaptophysin immunoreactivity was observed in BDNF +/+ and −/− mice in OHC soma (OHC, vertical arrows), implicating a BDNF-independent existence of synaptophysin-immunopositive presynaptic specializations in outer hair cells. In control mice at P6, synaptophysin immunoreactivity was restricted to fibers below outer hair cells (BDNF +/+,P6, OHC, vertical arrows), indicating the loss of synaptophysin-immunopositive presynaptic specializations in outer hair cells and the appearance of synaptophysin immunoreactivity in MOC efferents. In BDNF −/− mice, the MOC fiber population was absent at P2 and P6 (BDNF −/−,P2, P6, diagonal arrow), whereas synaptophysin-immunoreactive presynaptic specializations persisted in outer hair cells at both ages (P2,P6, vertical arrows). At P17, presynaptic specializations were lost in BDNF −/− mutants, whereas MOC efferents were still retarded in comparison to controls (compare diagonal arrows below outer hair cells in both specimens at P17). The experiment was repeated in quadruplicate with similar results.Arrowheads, IHC. Scale bar, 20 μm.

Fig. 7.

Afferent innervation of outer hair cells in the medial turn of control and BDNF gene-deleted mice analyzed by immunohistochemistry. GluR2/3-immunopositive fibers, presumptive afferent type I collaterals, were observed below outer hair cells in BDNF +/+ mice at P2 but not at P6 (BDNF +/+,GluR2/3, OHC, diagonal arrows). These GluR2/3-immunoreactive fibers were absent in BDNF −/− mice at both ages (BDNF −/−,GluR2/3, diagonal arrows). Anti-NF-200 stained presumptive afferent type II projections to OHCs in BDNF +/+ mice at P6 and P13 (BDNF +/+, NF-200,OHC, diagonal arrows), whereas BDNF −/− mice lacked this fiber population at both postnatal ages (BDNF −/−, NF-200, OHC,diagonal arrows). The experiment was repeated in triplicate with similar results. Arrowheads, IHC. Scale bar, 20 μm.

Fig. 8.

Innervation of inner hair cells in control and BDNF gene-deleted mice in the midbasal turn at P4 and P15 analyzed by immunohistochemistry. A, Staining of the immature synaptophysin-immunopositive efferent innervation at P4. Note the close localization of the efferent projections at the base of the IHCs in both mice specimens (compare the distance of thearrowhead and asterisk in BDNF +/+ and −/− mice at P4). B, Immunostaining of the afferent and efferent fibers by double-labeling at P15. GluR4-immunopositive afferent nerve endings were observed at the base of inner hair cell of BDNF +/+ and −/− mice (GluR4, filled arrowheads). In BDNF +/+ mice, GAP-43-immunopositive presumptive LOC terminals (GAP-43, open arrowheads) were localized in a distance to the base of the inner hair cell (filled arrowheads). In contrast to control, in BDNF −/− mice, GAP-43-immunopositive presumptive LOC terminals (GAP-43, open arrowheads) were observed at the hair cell base (filled arrowheads), indicating the persistence of axosomatic LOC synapses in BDNF −/− mice. Note the distance between the twoarrowheads in BDNF +/+ and −/− mice.Asterisks mark the hair cell nucleus. The experiment was repeated in triplicate with similar results. C, Comparison of efferent innervation of inner hair cells in control and BDNF gene-deleted mice at P15 by laser scanning confocal microscopy using an anti-synaptophysin antibody. In BDNF +/+ mice, a scattered elongated synaptophysin-immunopositive pattern was observed below the inner hair cell base (filled arrowheads), whereas in BDNF −/− the synaptophysin staining was concentrated at the base of inner hair cell (filled arrowheads). Differences in colors from blue to greento red mirror an intensity gradient of immunoreactivity.Asterisks mark the hair cell nucleus. Scale bars, 10 μm.